Helicobacter pylori, a gram-negative, microaerophilic spiral-shaped bacterium is the most frequently cited etiologic agent of human gastritis and peptic ulceration. This species, whose niche is highly restricted to the gastric mucosa of humans, has adopted a strategy of survival that includes synthesis of urease as its most abundant cellular protein. This enzyme hydrolyzes urea, releasing ammonia which allows colonization of this acid-sensitive organism at low gastric pH. In the previous funding period, we focused on factors that contribute to synthesis of a catalytically active urease. A rough topological model for the insertion of NixA, the high affinity nickel transport protein, into the cytoplasmic membrane has been established and we have identified 12 amino acid residues within the membrane domain that are critical for transport function. Three new urease-modulating factors including F1hA (flagellar biosynthesis/regulatory protein), Lpp (lipoprotein), and Hel (helicase) have been identified using a strategy similar to that used to isolate NixA. Glutamine synthetase which uses ammonia, the product of urea hydrolysis for production of glutamine from glutamate, has been characterized and demonstrated as essential for H. pylori survival. The role of Hpn, the histidine-rich protein, in bismuth sensitivity has also been elucidated. We postulate that NixA and other newly identified proteins are necessary for full activation of H. pylori urease. Since urea hydrolysis is 100 percent-dependent on nickel incorporation into urease, understanding this process could uncover targets for intervention. To address these research areas, we propose to use molecular genetic techniques, protein biochemistry, and bacterial physiology methodology: 1) To determine the mechanism by which urease activity is modulated. 2) To determine the fine structure of NixA, the mechanism of its gene regulation, and its contribution to virulence. 3) To determine the gene products that mediate transport of nickel ions across the inner and outer membranes of H. pylori.